PROJECT SUMMARY This proposal is focused on developing a vaccine for prevention of Powassan virus (POWV)- associated disease. POWV is a tick-borne flavivirus currently endemic to North America, primarily in the northeastern and north- central regions of the United States and extending north into Canada. Due to the expanding range of Ixodid tick vectors, POWV has the potential to become an emerging pathogen and public health threat. In humans, POWV is a neurotropic virus that can lead to severe, sometimes fatal encephalitis and meningitis. We propose to develop a vaccine based on non-replicating virus-like particle (VLP) antigens produced by expression of the viral pre-membrane (prM) and envelope (E) proteins in cultured cells followed by purification of VLP from the culture supernatant. VLPs will be paired with one of several novel vaccine adjuvants that stimulate specific pattern recognition receptors (PRRs). Each antigen/ adjuvant pairing will be tested in mice to quantify elicitation of neutralizing antibodies and POWV-specific T cells. We will also characterize B- and T-cell phenotypes in detail to determine how the vaccine-driven response compares to infection with intact POWV. Similarly to humans, POWV infection of wild-type laboratory mice results in lethal infection of the brain and CNS. Therefore, we will also evaluate vaccine efficacy in a challenge model of POWV infection in mice. Cohorts of mice vaccinated with individual antigen/ adjuvant pairings will be challenged with a lethal dose of POWV. These cohorts will be evaluated for increased survival, as well as, for reduction of viral load in tissues at multiple times post infection. Upon completion of this project, we expect to have developed an optimally formulated POWV vaccine which could move forward to clinical testing and be rapidly mobilized in the event of POWV emergence.